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01.12.2018 | Research article | Ausgabe 1/2018 Open Access

BMC Cancer 1/2018

An oncogenic mutant of RHEB, RHEB Y35N, exhibits an altered interaction with BRAF resulting in cancer transformation

Zeitschrift:
BMC Cancer > Ausgabe 1/2018
Autoren:
Jeffrey J. Heard, Ivy Phung, Mark I. Potes, Fuyuhiko Tamanoi
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12885-017-3938-5) contains supplementary material, which is available to authorized users.

Abstract

Background

RHEB is a unique member of the RAS superfamily of small GTPases expressed in all tissues and conserved from yeast to humans. Early studies on RHEB indicated a possible RHEB-RAF interaction, but this has not been fully explored. Recent work on cancer genome databases has revealed a reoccurring mutation in RHEB at the Tyr35 position, and a recent study points to the oncogenic potential of this mutant that involves activation of RAF/MEK/ERK signaling. These developments prompted us to reassess the significance of RHEB effect on RAF, and to compare mutant and wild type RHEB.

Methods

To study RHEB-RAF interaction, and the effect of the Y35N mutation on this interaction, we used transfection, immunoprecipitation, and Western blotting techniques. We generated cell lines stably expressing RHEB WT, RHEB Y35N, and KRAS G12V, and monitored cellular transforming properties through cell proliferation, anchorage independent growth, cell cycle analysis, and foci formation assays.

Results

We observe a strong interaction between RHEB and BRAF, but not with CRAF. This interaction is dependent on an intact RHEB effector domain and RHEB-GTP loading status. RHEB overexpression decreases RAF activation of the RAF/MEK/ERK pathway and RHEB knockdown results in an increase in RAF/MEK/ERK activation. RHEB Y35N mutation has decreased interaction with BRAF, and RHEB Y35N cells exhibit greater BRAF/CRAF heterodimerization resulting in increased RAF/MEK/ERK signaling. This leads to cancer transformation of RHEB Y35N stably expressing cell lines, similar to KRAS G12 V expressing cell lines.

Conclusions

RHEB interaction with BRAF is crucial for inhibiting RAF/MEK/ERK signaling. The RHEB Y35N mutant sustains RAF/MEK/ERK signaling due to a decreased interaction with BRAF, leading to increased BRAF/CRAF heterodimerization. RHEB Y35N expressing cells undergo cancer transformation due to decreased interaction between RHEB and BRAF resulting in overactive RAF/MEK/ERK signaling. Taken together with the previously established function of RHEB to activate mTORC1 signaling, it appears that RHEB performs a dual function; one is to suppress the RAF/MEK/ERK signaling and the other is to activate mTORC1 signaling.
Zusatzmaterial
Additional file 1: Figure S2. RHEB Y35N Exhibits Decreased Binding to BRAF. Cell lysates were collected from NIH 3T3 cell lines stably expressing FLAG-RHEB WT or FLAG-RHEB Y35N. Immunoprecipitation of endogenous BRAF was performed from these lysates. Western blots against BRAF and FLAG are shown. The cell line used for BRAF IP is indicated above the figure as WT (RHEB WT) or Y35N (RHEB Y35N) (PDF 161 kb)
12885_2017_3938_MOESM1_ESM.pdf
Additional file 2: Figure S3. Flow Cytometry Data for Cell Cycle Analysis. NIH 3T3 cell lines stably expressing FLAG-RHEB WT or FLAG-RHEB Y35N were grown for 2 days with serum (normal growth, top row) or without serum (serum starved, bottom row). Cells were then fixed, treated with RNase A to remove RNA, and incubated with propidium iodide (PI) to dye DNA. Cells were grouped into cell cycle stage based on PI intensity measured using flow cytometry. Flow cytometry statistics for each sample is shown to the right of each graph (PDF 434 kb)
12885_2017_3938_MOESM2_ESM.pdf
Additional file 3: Figure S1. RHEB Y35N Does not Exhibit Increased Binding to AMPK. A) RHEB WT, T38A, and Y35N mutants were transiently transfected and expressed in HEK 293T cells, cell lysates were collected, and immunoprecipitation for each was carried out. These results show a Western blot for AMPKα and FLAG from those samples. An effector domain mutant, RHEB T38A, did not bind AMPK demonstrating that AMPK is a relevant effector of RHEB (PDF 154 kb)
12885_2017_3938_MOESM3_ESM.pdf
Literatur
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